The cryoprotective effect of Guar gum co-supplemented with ethylene glycol and glycerol in Simmental bull semen.

Sperm cryopreservation often reduces sperm quality by forming of intra- and extracellular ice crystals. Various compounds widely used to counteract this effect. The guar gum was considered as an extracellular cryoprotective substance. The present study evaluated the impact of the co-supplementation of guar gum with ethylene glycol or glycerol in the cryopreservation of bull sperm. Four ejaculates from 4 bulls were pooled and divided into ten groups consisting of 4 controls (glycerol 6%, ethylene glycol 6%, glycerol 3.5%, and ethylene glycol 3.5%, and six treatment groups including guar gum in 0.001% and 0.002% alone and or co-supplemented either with 3.5% glycerol or 3.5% ethylene glycol and frozen in liquid nitrogen. The sperm motility, viability, plasma membrane and DNA integrity, apoptotic-like changes, antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities evaluated. The groups contained 3.5% glycerol + 0.001% guar gum, 3.5% ethylene glycol + 0.001% guar gum, and 0.001% guar gum alone showed higher values for live sperm, antioxidant enzymes, membrane integrity, mitochondrial membrane potential (MMP), fertilization, cleavage, and blastocyst rates; and lower values for apoptotic-like changes, H2O2 level, and DNA damage than the control groups. In conclusion, adding guar gum to the bull sperm diluent either alone or combined with glycerol or ethylene glycol ameliorated sperm viability and kinematic parameters and antioxidant capacity while reducing DNA damage and apoptotic-like changes. Guar gum also has improved embryo development. Due to its cost-effectiveness and physicochemical properties, guar gum is a promising supplement for bull sperm cryopreservation.

Exploring the feasibility of cryopreserving larvae of the common cockle (Cerastoderma edule) for hatchery production.

The decline of natural populations of the common cockle (Cerastoderma edule) through the European coast is posing a threat to local small-scale fisheries. These declines are primarily attributed to the prevalence of several pathogens and the disseminated neoplasia in cockle populations. The institution of a biobank of cryopreserved larvae could enhance hatchery production and help the restocking. The present work aimed at the development of a cryopreservation protocol for larvae of the common cockle using the mollusk cryopreservation protocols designed in our laboratory. Toxicity bioassays and short-term cryopreservation experiments were performed for protocol optimization according with cellular tolerance. Once settled, the viability of cryopreserved larvae was studied long term. Toxicity tests evidenced high tolerance of larvae against detrimental effects of Cryoprotecting Agents (CPAs). Cryopreservation of 48 h-old D-larva showed a 100% survival when increasing the equilibrium time from 15 to 60 min and using Propylene-Glycol (PG) + 0.4 M Trehalose (TRE) in Filtered Sea Water (FSW) and 60 min of exposure to CPA solution before slow-cooling. However, when cryopreserving the older larvae, the variation in equilibrium times hardly showed any effect but 10% Ethylene-Glycol (EG) + 0.4 M TRE and 60 min of exposure yielded the best relative survivorship (100%). Cryopreservation caused a significant delay on the growth rate of the latest larval stage. However, cryopreserved larvae survived to day 4-6, while 30 ± 12.17% of control larvae developed into pediveliger stage, of which 50% settled and transformed into juvenile cockles. These results demonstrated the role of the cell-type specificity in cryopreservation and highlight the importance of studying potential long-term effects of this tool to ensure the viability of the protocols.

Effect of cryopreservation on post-thaw motility and viability of Grey mullet, Mugil cephalus sperm (Linnaeus, 1758).

In this study, we optimized a simple method of cryopreservation for Mugil cephalus sperm based on post-thaw motility and viability. A series of experiments were conducted by changing the extender, cryoprotectant and freezing height above the liquid nitrogen (LN) surface. First, we carried out the cryopreservation using the extender V2E and cryoprotective agents (CPAs) namely, propylene glycol (PG), methanol (MeOH), glycerol (GLY), ethylene glycol (EG), dimethylsulfoxide (Me2SO) and dimethylacetamide (DMA) at a final concentration of 5% and 10%. We found that 10% of GLY, EG and Me2SO were more suitable compared to other CPAs. Then, different freezing heights (6, 8, 10 and 12 cm) above the LN surface were experimented with extender V2E and optimized CPAs. Then, 0.3 M of glucose, sucrose and trehalose were tested as extender along with optimized CPAs and freezing height. Additionally, the effect of fast-rate freezing and storage days (7, 30 and 180) on post-thaw sperm quality was documented using the factors optimized in earlier experiments. For all experiments, the fresh sperm was diluted at a ratio of 1:1 with cryomedium (CPA + extender), loaded into cryovials (2.0 mL) and frozen. The cryopreserved sperm was thawed at 30 °C for 90-120 s and their quality was evaluated. Among the experimented factors, sperm diluted in cryomedium (0.3 M glucose + 10% EG) and frozen at 4 cm above the LN surface registered significantly (P < 0.05) highest post-thaw motility (73 ± 2%) and (71 ± 1%) viability. Fast-rate freezing has resulted in lower (about 30%) post-thaw motility and viability of sperm. The storage days (7, 30 and 180) did not have a significant effect on post-thaw sperm quality. Overall results show that using the factors optimized through this study, high-quality sperm can be obtained after cryopreservation.

Pyrrosia petiolosa Extract Ameliorates Ethylene Glycol-Induced Urolithiasis in Rats by Inhibiting Oxidative Stress and Inflammatory Response.

Objective: To study the therapeutic effect and mechanism of Pyrrosia petiolosa (P. petiolosa) extract on ethylene glycol- (EG-) induced urolithiasis in rats. Methods: Thirty SD male rats were randomly divided into five groups (n = 6): control group, EG group, and P. petiolosa group (25 mg/kg, 50 mg/kg group, and 100 mg/kg). Biochemical testing was adopted for measuring the blood and urine parameters, as well as the level of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde acid (MDA) in kidney tissues. HE staining and ELISA were utilized to observe the histopathological changes and detect the level of IL-1ß, IL-6, MCP-1, and TNF-α in the kidney tissue, respectively. And western blot was performed for checking NOX2, NOX4, TGF-ß1, p-Smad3, Smad3, p-Smad2, and Smad2 protein expression level in kidney tissues. Results: EG could significantly increase the level of blood urea nitrogen, creatinine, and Na in serum and 24-hour urinary protein, oxalate, uric acid, creatinine, calcium, and phosphorus in urine and decreased the urine volume in rats. Whereas P. petiolosa extract was able to greatly decrease the level of related parameters in serum and urine in a dose-dependent manner, but did not affect the urine pH. In addition, P. petiolosa extract notably ameliorated EG-induced renal tissue injury. Compared with the EG group, P. petiolosa extract markedly raised the level of SOD and GSH and decreased the MDA level and the expression of NOX2 and NOX4 in the kidney tissue. Moreover, P. petiolosa extract also lowered the level of IL-1ß, IL-6, MCP-1, and TNF-α in EG-stimulated kidney tissue and inhibited the protein level of EG-induced TGF-ß1, p-Smad3, and p-Smad2 in a concentration-dependent manner. Conclusion: P. petiolosa extract can improve EG-induced urolithiasis in rats by inhibiting oxidative stress, inflammatory response, and the activation of TGF-ß pathway.

Nanogel-mediated drug delivery system for anticancer agent: pH stimuli responsive poly(ethylene glycol/acrylic acid) nanogel prepared by gamma irradiation.

The popularity of nanogel as nano drug carrier lies in its adjustable physical properties, and the ability to encapsulate drug particles with improved properties is being developed to meet the diverse pH-sensitive nanogel for anticancer agent. Monitoring pH has been identified as an important diagnostic element during the treatment process. A pH-sensitive nanogel consisting of (PEG/PMAc) in the ratio of (50:50%) hasbeen cross-linkedby γ-irradiation techniques at an irradiation dose of 5 kGy. Compound 4 and its nanogel 5 were synthesized and assessed for their anticancer effects against HepG2, A549, MCF-7 and HCT-116 as dual VEGFR-2 and EGFR tyrosine kinases inhibitors. The molecular design was performed to investigate the binding mode of compound 4 with VEGFR-2 and EGFR receptors. Our compound 5 in nanogel showed enhanced anticancer activities against the four tested cancer cell lines and also showed higher inhibition activities against VEGFR-2 and EGFRT790M kinases than the derivative 4. Finally, our derivative 4 showed good in silico calculated ADMET profile. It was expected to show good GIT absorption in human, lower CNS side effects, no hepatotoxic actions and higher acute and oral chronic toxic doses in comparing to sorafenib and erlotinib. The obtained results showed that, our compound could be useful as a template for future design, optimization, adaptation and investigation to produce more potent and selective dual VEGFR-2/EGFRT790M inhibitors with higher anticancer activity.

Ram epididymal sperm frozen in an extender containing ethylene glycol have higher post-thaw longevity and in vitro fertility.

BACKGROUND: Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of spermatozoa could be maintained for a longer period after thawing is of great practical value. OBJECTIVES: To optimize freezing and thawing protocol for ram epididymal sperm using either ethylene glycol (EG) or glycerol (GLY) as cryoprotectants (CPAs). Then, to evaluate the post-thaw longevity and in vitro fertility of spermatozoa that were frozen and thawed according to the optimized protocol. MATERIALS AND METHODS: At first, an optimum protocol for freezing and thawing sperm using EG or GLY were investigated, and the next experiments were performed using the spermatozoa that had been frozen and thawed according to the optimized protocol for each CPA. In the next experiments, frozen-thawed and fresh sperm were diluted in an isotonic culture medium and subsequently incubated at 39°C for 4 h. The motility characteristics and functional membrane integrity (FMI) of spermatozoa were evaluated after thawing, after dilution (t0 ), and after incubation (t4 ). The in vitro fertility of the spermatozoa was assessed at t0 and t4 . RESULTS: For both CPAs, the highest motility parameters and FMI was found for spermatozoa frozen at 3 cm above LN2 and thawed at 50 and 65°C (P < 0.05). In comparison to the spermatozoa of GLY group, the spermatozoa of the EG group had higher total and progressive motility at t0 , as well as higher FMI, total and progressive motility, and linearity at t4 (P < 0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t0 (P < 0.05). Incubation treatment increased the fertility of fresh sperm while decreased the fertility of frozen-thawed sperm, and this decline was more severe in GLY than in the EG group. CONCLUSION: Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes†.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Conjugate of Thiol and Guanidyl Units with Oligoethylene Glycol Linkage for Manipulation of Oxidative Protein Folding.

Oxidative protein folding is a biological process to obtain a native conformation of a protein through disulfide-bond formation between cysteine residues. In a cell, disulfide-catalysts such as protein disulfide isomerase promote the oxidative protein folding. Inspired by the active sites of the disulfide-catalysts, synthetic redox-active thiol compounds have been developed, which have shown significant promotion of the folding processes. In our previous study, coupling effects of a thiol group and guanidyl unit on the folding promotion were reported. Herein, we investigated the influences of a spacer between the thiol group and guanidyl unit. A conjugate between thiol and guanidyl units with a diethylene glycol spacer (GdnDEG-SH) showed lower folding promotion effect compared to the thiol-guanidyl conjugate without the spacer (GdnSH). Lower acidity and a more reductive property of the thiol group of GdnDEG-SH compared to those of GdnSH likely resulted in the reduced efficiency of the folding promotion. Thus, the spacer between the thiol and guanidyl groups is critical for the promotion of oxidative protein folding.

Development of a method to cryopreserve Greenshell mussel™ (Perna canaliculus) veliger larvae.

Cryopreservation of larvae of Greenshell™ mussel Perna canaliculus, the most cultivated species in New Zealand, can provide flexibility for selective breeding programmes and enhance its global production. In this study, we set out to develop a reliable protocol for freezing D-stage larvae of Greenshell™ mussels that ensured long-term survival for successful rearing of thawed larvae in the hatchery. The effects of different combinations of cryoprotecting agents (CPA), varying CPA equilibration times, larval concentrations per straw as well as different larval development stages (48 h vs 72 h old) were evaluated by assessing the behavioural response (swimming activity, algal consumption), shell size and survival of larvae, up to 4 days post-thawing. The protocol yielding the best larval performances was a combination of the following CPA (final concentrations): 14% ethylene-glycol (EG) + 0.6 M trehalose (TRE) + 1% polyvinyl-pyrrolidone (PVP), prepared with Milli-Q water. Stocking densities ranging from 50,000 to 150,000 larvae per straw (0.25 mL) and a 20 min equilibration time gave the best results, while no significant differences in fitness were found between larvae cryopreserved at 48 h nor 72 h-old. Using the improved cryopreservation protocol, over 50% of previously cryopreserved D-larvae were able to survive after 4 days of rearing, compared with 65% in the unfrozen control. More importantly, about one third of thawed larvae were able to swim and feed, and to potentially develop further. These findings contribute to enhance the selective breeding programmes for this species.

A fully synthetic 6-aza-artemisinin bearing an amphiphilic chain generates aggregates and exhibits anti-cancer activities.

Installation of a nitrogen at the C6 position of artemisinin facilitates the addition of a functional unit on the cyclohexane moiety (C-ring). In this study, conjugation of an amphiphilic chain, composed of sequentially connected hydrophilic oligoethylene glycol, hydrophobic alkyl chain, urea, and 4,4'-disubstituted biphenyl linker, imparted self-assembling properties. The fully synthetic mid-molecular weight 6-aza-artemisinin 6 bearing the amphiphilic moiety formed aggregates (approx. 200 nm) at ambient temperature and exhibited increased in vitro anti-cancer activities compared to the N-benzylated aza-artemisinin 5.

Variation in sperm cryosurvival is not modified by replacing the cryoprotectant glycerol with ethylene glycol in bulls.

Breed and sire differences in sperm cryosurvival have been noted, with negative implications for sperm cryobanking and assisted reproduction programmes. This study hypothesized that these differences could be modified by using lower molecular weight cryoprotectants. Therefore, the effect of replacing glycerol (GLY) with ethylene glycol (EG) on differential cryosurvival of semen from two Sanga cattle breeds (Mashona vs. Tuli) was determined. Three to five ejaculates were collected from each of ten bulls (3-8 years) by electro-ejaculation, diluted in three Tris-egg yolk extenders (Triladyl® , 7% GLY-based and 7% EG-based) and evaluated for sperm motility, viability and morphology at three time periods (fresh - 0 hr, pre-freeze - 4 hr and post-thaw). Tuli bulls produced larger (11.8 ± 0.31 ml vs. 8.5 ± 0.38 ml) and more concentrated ejaculates of lower fresh semen quality. Breeds differed across time for motility and morphology, but not viability. Mashona bull semen had significantly higher motility and normal morphology values at each sampling time. Bulls classified as poor freezers had lower concentration (0.70 ± 0.09 × 109  sperm/ml vs. 1.37 ± 0.10 × 109  sperm/ml), sperm motility index (SMI, 35.0 ± 3.4 % vs. 67.8 ± 2.1 %) and viability (69.7 ± 1.1 % vs. 75.7 ± 1.0 %) compared to good freezers. Maintenance of semen quality by GLY and EG did not differ between breeds, poor and good freezers, or age groups. The interaction breed by extender across time did not reach statistical significance for all variables. The study revealed that bull and breed variation in sperm quality and cryosurvival is not modified by replacing GLY with EG, suggesting that cryostress tolerance of sperm may be under control of mechanisms other than differential response to GLY cytotoxicity.

Long-term (24h) cooling of ovarian fragments in the presence of permeable cryoprotectants prior to freezing: Two unsuccesful IVF-cycles and spontaneous pregnancy with baby born after re-transplantation.

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 °C for 22-24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at -9 °C, cooling from -9 to -34 °C at a rate of -0.3 °C/min and plunging at -34 °C into liquid nitrogen. After thawing in a 100 °C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 ± 1.9% and 96.4 ± 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months after the auto-transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Long-term (24 h) cooling of ovarian tissue to 5 °C before cryopreservation in the presence of permeable cryoprotectants simplifies the protocol of cryopreservation and has a tendency of increasing of the cells viability after thawing.

Formation of a structurally-stable conformation by the intrinsically disordered MYC:TRRAP complex.

Our primary goal is to therapeutically target the oncogenic transcription factor MYC to stop tumor growth and cancer progression. Here, we report aspects of the biophysical states of the MYC protein and its interaction with one of the best-characterized MYC cofactors, TRansactivation/tRansformation-domain Associated Protein (TRRAP). The MYC:TRRAP interaction is critical for MYC function in promoting cancer. The interaction between MYC and TRRAP occurs at a precise region in the MYC protein, called MYC Homology Box 2 (MB2), which is central to the MYC transactivation domain (TAD). Although the MYC TAD is inherently disordered, this report suggests that MB2 may acquire a defined structure when complexed with TRRAP which could be exploited for the investigation of inhibitors of MYC function by preventing this protein-protein interaction (PPI). The MYC TAD, and in particular the MB2 motif, is unique and invariant in evolution, suggesting that MB2 is an ideal site for inhibiting MYC function.

Effect of the Hydrophilic-Hydrophobic Balance of Antigen-Loaded Peptide Nanofibers on Their Cellular Uptake, Cellular Toxicity, and Immune Stimulatory Properties.

Recently, nanofibers (NFs) formed from antigenic peptides conjugated to ß-sheet-forming peptides have attracted much attention as a new generation of vaccines. However, studies describing how the hydrophilic-hydrophobic balance of NF components affects cellular interactions of NFs are limited. In this report, three different NFs were prepared by self-assembly of ß-sheet-forming peptides conjugated with model antigenic peptides (SIINFEKL) from ovalbumin and hydrophilic oligo-ethylene glycol (EG) of differing chain lengths (6-, 12- and 24-mer) to investigate the effect of EG length of antigen-loaded NFs on their cellular uptake, cytotoxicity, and dendritic cell (DC)-stimulation ability. We used an immortal DC line, termed JAWS II, derived from bone marrow-derived DCs of a C57BL/6 p53-knockout mouse. The uptake of NFs, consisting of the EG 12-mer by DCs, was the most effective and activated DC without exhibiting significant cytotoxicity. Increasing the EG chain length significantly reduced cellular entry and DC activation by NFs. Conversely, shortening the EG chain enhanced DC activation but increased toxicity and impaired water-dispersibility, resulting in low cellular uptake. These results show that the interaction of antigen-loaded NFs with cells can be tuned by the EG length, which provides useful design guidelines for the development of effective NF-based vaccines.

Cryoprotectant toxicity in Caenorhabditis elegans.

We have looked at the effects of the cryoprotectant M22 upon viability in the model organism C. elegans. M22 is a well-known vitrification solution which has been successfully used in the laboratory to preserve organs destined for transplantation. M22 reduces survival of C. elegans in a concentration-dependent manner. M22 at concentrations of 10% (v/v) or higher inhibits progeny production and development. A few mutants in the ILS (insulin-like signaling) pathway of C. elegans are more resistant to the toxic effect of M22 compared to wild-type worms. Afatinib, an anti-cancer drug, protects against M22 toxicity. Afatinib by itself does not increase longevity.

Changing expression profiles of long non-coding RNAs, mRNAs and circular RNAs in ethylene glycol-induced kidney calculi rats.

BACKGROUND: To explore long non-coding RNA (lncRNA), mRNA and circular RNA (circRNA) expression profiles and their biological functions in the pathogenesis of kidney stones in ethylene glycol-induced urolithiasis rats. RESULTS: The expression of 1440 lncRNAs, 2455 mRNAs and 145 circRNAs was altered in the kidneys of urolithiasis rats. GO and KEGG biological pathway analysis were performed to predict the functions of differentially expressed lncRNAs, circRNAs and co-expressed potential targeting genes. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were constructed based on correlation analysis between differentially expressed RNAs. mRNAs coexpressed with lncRNAs were involved in many kidney diseases, e.g., Ephb6 was associated with the reabsorption ability of the kidney. Arl5b was associated with the dynamic changes in the podocyte foot process in podocyte injury. miRNAs co-expressed with circRNAs, such as rno-miR-138-5p and rno-miR-672-5p, have been proven to be functional in hypercalciuria urolithiasis. CONCLUSION: The expression profile provided a systematic perspective on the potential functions of lncRNAs and circRNAs in the pathogenesis of kidney stones. Differentially expressed lncRNAs and circRNAs might serve as treatment targets for kidney stones.

Cat ovarian follicle ultrastructure after cryopreservation with ethylene glycol and dimethyl sulfoxide.

Ovarian tissue cryopreservation is a promising technique for fertility maintenance. The aim of this study was to compare the morphology of domestic cat ovarian follicles after tissue cryopreservation with ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). Ovaries from healthy adult cats undergoing elective ovariohysterectomy were used. Eight fragments were obtained from each pair of ovaries: two were used as fresh controls; three were submitted to fresh perfusion toxicity test and perfused with M199, 10% fetal calf serum and 0.4% sucrose containing Me2SO 1.5 M, EG 1.5 M or Me2SO 0.75 M + EG 0.75 M; and the remaining three fragments were perfused as described and submitted to slow freezing. After 45 days of cryopreservation, the samples were thawed, fixed and processed for light and transmission electron microscopy (TEM). The percentages of morphologically normal follicles identified by light microscopy were higher in the control group (94.45%) in comparison to the frozen groups (80.56% with EG, 78.7% with Me2SO and 75.87% with EG + Me2SO). The fresh perfused tissue showed no statistical difference compared to control or frozen samples. The TEM analysis showed less damage in the ultrastructure of follicles from the Me2SO group in comparison with the EG and Me2SO + EG groups. According to the morphological analysis, 1.5 M Me2SO is the best cryoprotectant for cryopreservation of domestic cat ovarian tissue regarding the morphology of preantral follicles after thawing. Further studies regarding the viability of these follicles should be performed.

Effects of five cryoprotectants on proliferation and differentiation-related gene expression of frozen-thawed bovine calf testicular tissue.

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7-day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)-related genes, octamer-4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C-KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.

Inhibition of Autophagy Attenuated Ethylene Glycol Induced Crystals Deposition and Renal Injury in a Rat Model of Nephrolithiasis.

BACKGROUND/AIMS: Nephrolithiasis is a common and frequently occurring disease, its exact pathogenesis is remains unclear. Emerging data suggest that autophagy plays a vital role in the pathophysiological processes of kidney diseases. Therefore, this study was designed to investigate the potential role of autophagy in the formation of calcium oxalate (CaOx) kidney stones in rat model. METHODS: Thirty-two rats were randomly divided into four groups (eight rats/group): untreated control group, stone model group, rapamycin-treated group, chloroquine-treated group. Rat models of CaOx nephrolithiasis was administration of 0.75% ethylene glycol (EG) in their drinking water for 4 weeks. Western blot and transmission electron microscope (TEM) were used to detect the expression of autophagy related protein LC3-II, BECN1 and p62 and autophagic vacuoles respectively. Renal function was evaluated by measuring the levels of serum CRE and BUN. Renal tubular injury markers NGAL and Kim-1 was determined by ELISA kits. Von Kossa staining was used to assess crystal deposits and histological tissue injury. TUNEL staining was employed to assess apoptosis of the renal tubular cell. RESULTS: Compare with the controls, the expression of autophagy related protein LC3-II, BECN1 and number of autophagic vacuoles were increased significantly, whereas the p62 protein level was decreased in the stone model group. The levels of apoptosis, serum CRE and BUN, NGAL and Kim-1 in the stone model group were increased compared with the control group and crystals deposition and renal injury were increased significantly. However, the levels of autophagy, kidney injury and crystal deposition were decreased by chloroquine but increased by rapamycin. CONCLUSION: These findings suggested that rats were administration of ethylene glycol could lead to the formation of CaOx nephrolithiasis and autophagy activation. Inhibiting autophagy could be an effective therapeutic approach for decreasing the formation of nephrolithiasis.

Ethylene Glycol and Dimethyl Sulfoxide Combination Reduces Cryoinjuries and Apoptotic Gene Expression in Vitrified Laying Hen Ovary.

Successful cryopreservation of avian gonads is important not only for avian breeding but is also crucial for preservation of species, especially of endangered birds. The aim of this study was to evaluate the effect of vitrification by several cryoprotectants on the ovarian tissues of laying hens. Ovarian tissues were randomly divided into six groups: control (nonvitrified: C), dehydrated using ethylene glycol (EG), dehydrated with propylene glycol (PROH), dehydrated using dimethyl sulfoxide (DMSO), and two combined groups, EG+DMSO and EG+PROH. The composition of vitrification solutions was as follows: EG group: V1 = 7.5% EG and V2 = 15% EG +0.5 M sucrose, DMSO group: V1 = 7.5% DMSO and V2 = 15% DMSO +0.5 M sucrose, PROH group: V1 = 7.5% PROH and V2 = 15% PROH +0.5 M sucrose, EG+DMSO group: V1 = 7.5% EG +7.5% DMSO and V2 = 15% EG +15% DMSO +0.5 M sucrose and EG+PROH group: V1 = 7.5% EG +7.5% PROH and V2 = 15% EG +15% PROH +0.5 M sucrose. Ovarian tissues of each group were dehydrated for 10 minutes with V1 solution and 2 minutes with V2. Among the vitrified groups, intact primordial and primary follicles showed significant increase in EG+DMSO, but follicular attrition had the highest rate in the PROH group (p < 0.05). Immunohistochemical analysis showed that the percentage of active caspase 3-positive cells was lower (p < 0.05) when using EG+DMSO versus PROH. Further gene expression of caspase 3, 8, and 9 was highest in the PROH group (p < 0.05). Vitrification of ovaries of laying hens using EG+DMSO can afford effective protection of primordial and primary follicles during preservation and may therefore be successfully used for storing avian gonadal tissues.